Ovarian cancer is lethal because of near-universal development of resistance to platinum-based chemotherapy. Metabolic adaptations can play a pivotal role in therapy resistance. In this study, we aimed to identify key metabolic pathways that regulate platinum response and represent potential therapeutic targets. Transcriptomic and metabolomic analyses in cisplatin-sensitive and -resistant ovarian cancer cells identified enrichment of pyrimidine metabolism related to upregulated de novo pyrimidine synthesis. The 15N-glutamine flux analysis confirmed increased de novo pyrimidine synthesis in cisplatin-resistant cells. Targeting this pathway using brequinar (BRQ), an inhibitor of the key enzyme dihydroorotate dehydrogenase, decreased cell viability, delayed G2/M cell-cycle progression, and altered expression of genes related to mitochondrial electron transport in resistant cells. Under basal conditions, cisplatin-resistant cells had a lower oxygen consumption rate and spare respiratory capacity than cisplatin-sensitive cells. BRQ suppressed the oxygen consumption rate in both sensitive and resistant cells but only inhibited spare respiratory capacity in resistant cells. In cell line-derived and patient-derived xenograft models, BRQ attenuated the growth of cisplatin-resistant ovarian tumors and enhanced the inhibitory effects of carboplatin. Together, these results identify metabolic reprogramming in cisplatin-resistant ovarian cancer that induces an acquired dependency on de novo pyrimidine synthesis, which can be targeted to sensitize tumors to chemotherapy.Significance: De novo pyrimidine synthesis supports platinum resistance in ovarian cancer and can be targeted with DHODH inhibitors to suppress tumor growth, pointing to potential metabolic therapies for treating recurrent ovarian cancer.

Background Ovarian cancer (OC) depends on lipids as fuel for metastasis and growth. We previously showed that cisplatin resistant (Pt-R) OC cells uptake higher amounts of fatty acids (FAs) compared to sensitive (Pt-S) cells, a process which facilitates cancer cell survival under cisplatin-induced oxidative stress. Methods Isogenic pairs of Pt-S and Pt-R OC cell lines were cultured in low serum conditions supplemented with either 50 μM oleic acid (OA, unsaturated) or 50 μM palmitic acid (PA, saturated) and used for viability assays, RNA-Sequencing, and cell cycle analysis. The effects of an OA enriched diet were assessed in intraperitoneal ovarian xenografts. The FABP inhibitor BMS-309403 was used to block lipid import in vitro and in vivo. Results Pt-R cells were less viable than Pt-S cells under serum depletion and OA rescued starvation induced inhibition of cell proliferation, with more significant effects in Pt-R compared to Pt-S cells. RNA-sequencing showed that OA promoted upregulation of cell cycle-related pathways, including G2/M checkpoints, driven by the transcription factor E2F1. Supplementation with OA increased S- and G2/M phase cell populations in both Pt-S and Pt-R cells (p<0.05) and E2F1 inhibition reduced OA-induced cell proliferation. An OA enriched diet promoted the growth and peritoneal dissemination of Pt-R ovarian xenografts. When co-cultured with adipocytes, Pt-R cells expressed higher levels of FA transporter proteins FABP4 and CD36 compared to sensitive cells and FABP4 expression was upregulated in paired metastatic and recurrent vs. primary human ovarian tumors (p<0.05). An FABP inhibitor sensitized OC cells to cisplatin and suppressed the in vivo growth of Pt-R xenografts and patient derived xenografts. Conclusions Pt-R OC cells harbor heightened dependence on unsaturated FAs compared to Pt-S cells and upregulate key transporters to increase FAs uptake. OA supports the proliferation of Pt-R cells in vitro and in vivo and a combination of carboplatin and FABP4 inhibitor reduces OC growth in vivo. These findings suggest that lipid composition may influence therapeutic response and raise important considerations for dietary guidance in patients with cancer.

Ovarian cancer (OC) depends on fat as fuel for metastasis and growth. We have previously demonstrated that cisplatin resistant (Pt-R) OC cells uptake higher amounts of fatty acids (FAs) compared to sensitive (Pt-S) cells. Here, we determined the mechanism through which FAs support cancer survival. We used two isogenic OC cell lines pairs, sensitive and resistant to cisplatin: OVCAR5 Pt-S (IC50 = 2.881mM) vs Pt-R (IC50 = 6.831 mM) and PEO1 (IC50 = 4.901 mM) vs PEO4 (IC50 = 8.285 mM). Cells were cultured under low serum conditions, supplemented with 50 µM oleic (OA) or 50 µM palmitic acid (PA), and used for viability assays, RNA-Seq, cell cycle, and cell death analysis. Quantitative RT-PCR measured expression of key FAs transporters in Pt-S and Pt-R cells. Immunohistochemistry (IHC) assessed expression of FABP4 in primary and recurrent human tumors. The FABP inhibitor BMS-309403 was used to pharmacologically inhibit the FA transporters in vitro and in vivo. Pt-R cells rely more on unsaturated FAs than Pt-S cells, upregulating transporters for increased FA uptake. Cell viability assays showed Pt-R cells were less viable under serum depletion, with oleic acid (OA, unsaturated) promoting proliferation of both OC cell lines. OA rescued Pt-R cells significantly more than Pt-S cells: 268.4% vs 91.6% for OVCAR-5 and 38.1% vs 12.1% for PEO4 vs PEO1. OA also reduced apoptosis, as shown by Annexin V assays, and decreased Caspase-3 and -8 cleavage. RNA-Seq analysis linked OA-induced proliferation to upregulation of cell cycle-related pathways, including G2/M checkpoints, driven by E2F1. OA increased S- and G2/M phase populations (e.g., 6.95% to 11.6%; p<0.05), while E2F1 inhibition reduced OA-induced proliferation. Pt-R cells also expressed higher levels of transported proteins FABP4 and CD36. Inhibiting FABPs reduced Pt IC50 values (e.g., from 11.893 μM to 5.0415 μM in OVCAR5) and blocked ovarian cancer progression in vivo. IHC showed FABP4 overexpression in metastatic and recurrent tumors (n=21 paired tumors, p<0.05). Pt-R OC cells are more dependent on unsaturated FAs compared to Pt-S cells and upregulate key transporters to allow increased uptake of FAs. Inhibiting intracellular FA transport reduces OC progression in vivo. OA supports proliferation of Pt-R cells by rescuing them from apoptosis and by promoting cell cycle progression from G1 to S- and G2/M phases. Ana Maria Isac, Andres Valdivia, Guangyuan Zhao, Yinu Wang, Jian-Jun Wei, Sandra Orsulic, Daniela Matei. Unsaturated fatty acids promote cell cycle progression and proliferation of platinum resistant ovarian cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1536.

High grade serous ovarian cancer (OC) is lethal due to near universal development of resistance to platinum-based chemotherapy. We used cisplatin resistant (Cis-R) cells generated by repeated cisplatin treatment of sensitive (Cis-S) OC cell lines, transcriptomic and metabolomic analyses to identify key metabolic pathways associated with platinum resistance. In vivo experiments used intraperitoneal (ip) OC xenografts and patient derived xenografts (PDX). Pathway analysis of differentially abundant metabolites in Cis-R vs. Cis-S OVCAR5 and COV362 cells identified enrichment of pyrimidine metabolism (FDR<0.05) related to increased de novo pyrimidine synthesis. 15N-glutamine flux analysis confirmed increased de novo pyrimidine synthesis in Cis-R cells. Inhibition of this pathway using the DHODH inhibitor brequinar (BRQ) decreased cell viability and induced expression changes in genes of the mitochondrial electron transport in Cis-R compared with Cis-S cells. Oxygen consumption rate (OCR) measured with a Seahorse assay indicated lower (P<0.05) OCR (baseline, ATP-related, and maximal) and spare respiratory capacity (SRC) in Cis-R vs. Cis-S OVCAR5 cells. BRQ induced dose-related inhibition of OCR in both Cis-R and Cis-S cells. SRC was inhibited by BRQ only in Cis-R cells. In vivo, BRQ attenuated growth of ip Cis-R OC xenografts but did not alter Cis-S tumors. Tumor growth was also inhibited (p<0.01) by BRQ in a Cis-R PDX model. BRQ plus carboplatin enhanced (p<0.05) the inhibitory effects of carboplatin alone in ip Cis-R OC xenografts. Our results support that Cis-R cells acquire dependency on the de novo pyrimidine synthesis and maintain relatively low levels of mitochondrial oxidation, rendering Cis-R cells and tumors vulnerable to inhibitors of this pathway. Horacio Cardenas, Guangyuan Zhao, Ana Maria Isac, Daniela E. Matei. Metabolic dependency on de novo pyrimidine synthesis in platinum resistant ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3073.

The omentum is the primary site of metastasis for ovarian cancer (OC). Interactions between cancer cells and adipocytes drive an invasive and prometastatic phenotype. Here we studied cancer cell- adipocyte crosstalk by using a direct coculture model with immortalized human visceral nondiabetic pre-adipocytes (VNPADs) and OC cells. We demonstrated increased proliferation, invasiveness, and resistance to cisplatin of cocultured compared with monocultured OC cells. RNA sequencing of OC cells from coculture versus monoculture revealed significant transcriptomic changes, identifying over 200 differentially expressed genes common to OVCAR5 and OVCAR8 cell lines. Enriched pathways included PI3K/AKT and complement activation. Lipid transfer into OC cells from adipocytes induced upregulation of complement C3 and C5 proteins. Inhibiting C3 or C5 reversed the invasive phenotype and C3 knockdown reduced tumor progression in vivo. Increased C3 expression was observed in omental implants compared with primary ovarian tumors and C3 secretion was higher in OC ascites from high-BMI versus low-BMI patients. C3 upregulation in OC cells involved activation of the ATF4-mediated integrated stress response (ISR). Overall, adipocyte-cancer cell interactions promoted invasiveness and tumorigenesis via lipid transfer, activating the ISR, and upregulating complement proteins C3 and C5.

Tissue transglutaminase (TG2) is a multifunctional protein with roles in multiple diseases including cancer. It was found overexpressed in many solid tumors in the past two decades. In ovarian cancer, it emerged as a potential therapeutic target due to its involvement in processes like metastasis and chemo- and radiotherapy resistance. Generally, TG2 expression in cancer cells has been extensively studied and linked to increased tumor progression. However, its role in the host was less studied. Our research explored the anti-tumor immune response using a TG2KO syngeneic ovarian cancer mouse model, as compared to wild-type animals. We observed decreased tumor burden and increased survival upon i.p. injection of ID8 mouse ovarian cancer cells in TG2KO mice, as compared to wild-type. In the absence of TG2 in the host, an increased infiltration of CD8+ T cells in ascites was evidenced by FACS, while myeloid cells were less present. The TG2KO CD8+ T cells showed increased activation and increased effector function. Moreover, these cells showed an increased cancer cell killing capacity. At the molecular level, this phenotype was supported by attenuated STAT3 phosphorylation. Cancer cells in ascites collected from TG2KO mice showed a gene signature corresponding to IFN-γ response. Overall, these data show decreased tumor progression and increased effector phenotype in CD8+ T cells when TG2 is absent in the host (Sima LE et al. (2021) Journal for ImmunoTherapy of Cancer 9, e002682). When looking to confirm these results in human OC tumor microarrays (TMAs), we found an inverse correlation between human stromal (but not tumor) TG2 expression and CD8+ T cells infiltration by IHC staining followed by StrataQuest image cytometry. Using Opal-based multiplex IHC staining, we identified TG2 in a subset of cancer associated fibroblasts (CAFs). TG2 was previously reported as a non-toxic druggable target involved in “outside-in” signaling downstream of integrins. Further, we used inhibitors developed towards TG2-fibronectin interaction (Sima LE et al. (2019) Mol Cancer Ther 18, 1057-1068) to evaluate how they affect the cancer cells-fibroblasts cross-talk. This treatment prevented the self-assembly of heterospheroids. This is relevant for the situation in the tumor microenvironment, where we found TG2-FN interaction present in both stroma and tumor areas. These results support the potential use of TG2 directed agents in ovarian cancer therapy, as we propose TG2 as a new immunomodulatory target. Our current efforts also include using FLASH radiation to sensitize cancer cells to immunotherapy. This research was supported by funding from the US Department of Veterans Affairs, Robert H Lurie Comprehensive Cancer Center, UEFISCDI (PN-III- P1-1.1-TE- 2019-0670; PN-III-P2-2.1-PED-2019-1543) and IFA (ELI-RO_10/2024).

BackgroundTumor heterogeneity is one of the key factors leading to chemo-resistance relapse. It remains unknown how resistant cancer cells influence sensitive cells during cohabitation and growth within a heterogenous tumors. The goal of our study was to identify driving factors that mediate the interactions between resistant and sensitive cancer cells and to determine the effects of cohabitation on both phenotypes.MethodsWe used isogenic ovarian cancer (OC) cell lines pairs, sensitive and resistant to platinum: OVCAR5 vs. OVCAR5 CisR and PE01 vs. PE04, respectively, to perform long term direct culture and to study the phenotypical changes of the interaction of these cells.ResultsLong term direct co-culture of sensitive and resistant OC cells promoted proliferation (p < 0.001) of sensitive cells and increased the proportion of cells in the G1 and S cell cycle phase in both PE01 and OVCAR5 cells. Direct co-culture led to a decrease in the IC50 to platinum in the cisplatin-sensitive cells (5.92 µM to 2.79 µM for PE01, and from 2.05 µM to 1.51 µM for OVCAR5). RNAseq analysis of co-cultured cells showed enrichment of Cell Cycle Control, Cyclins and Cell Cycle Regulation pathways. The transcription factor E2F1 was predicted as the main effector responsible for the transcriptomic changes in sensitive cells. Western blot and qRT-PCR confirmed upregulation of E2F1 in co-cultured vs monoculture. Furthermore, an E2F1 inhibitor reverted the increase in proliferation rate induced by co-culture to baseline levels.ConclusionOur data suggest that long term cohabitation of chemo-sensitive and -resistant cancer cells drive sensitive cells to a higher proliferative state, more responsive to platinum. Our results reveal an unexpected effect caused by direct interactions between cancer cells with different proliferative rates and levels of platinum resistance, modelling competition between cells in heterogeneous tumors.

Abstract Background: Ovarian cancer is a highly lethal gynecological malignancy and high-grade serous histology (HGSOC) accounts for the majority of cases. Platinum-based chemotherapy is the primary treatment. Notably, Black women face the highest mortality-to-incidence ratio across all ethnic groups. This study investigated transcriptomic and immunological differences in tumors from Black compared to NHW women that might explain the poor clinical outcomes observed within this patient cohort. Methods: We collected primary tumor specimens from 36 Black and 31 treatment-naïve NHW patients. After RNA isolation, RNA sequencing (RNA-seq) identified differentially expressed transcripts and Enrichr performed pathway enrichment studies. To confirm the observed gene expression differences, we employed quantitative reverse transcription PCR (qRT-PCR), western blotting, and multiplex immunohistochemistry (mIHC). Additionally, we conducted cell proliferation, colony formation, and cell viability assays to functionally validate potential target genes of interest. Results: Our findings revealed 277 genes with significant differential expression between Black and NHW patients (FDR-adjusted p-value < 0.05). Among these, 103 coding genes were up-regulated, while 81 coding genes were down-regulated in tumors from Black compared to NHW patients. Gene Ontology analyses of these significantly differentially expressed genes highlighted enriched pathways related to DNA damage response, including the insulin receptor (INSR) gene, p53/apoptosis signaling components such as Forkhead box proteins A1 (FOXA1) and FOXB1, as well as genes involved in the cholesterol/lipid modulation pathway, including Low-density lipoprotein (LDL) receptor and Stearoyl-CoA Desaturase (SCD). Notably, silencing INSR and FOXA1 enhanced sensitivity to platinum-based drugs and inhibited cell growth and colony formation. Furthermore, we identified differences in the proportions of key immune cell types between the two patient groups, with tumors from Black patients exhibiting a significantly lower proportion of CD4+ Naïve T-cells and CD4+ regulatory T-cells (Tregs). Conclusions: Overall, our study reveals significant differential gene expression patterns between HGSOC tumors from Black and NHW patients, as well as differences in the proportions of immune cell types. These discoveries provide valuable insights into the biological mechanisms underlying the disparities in outcomes observed between black and NHW patients afflicted with HGSOC. It is critical to further investigate how these biological differences affect clinical outcomes and treatment response in Black women. Citation Format: Hao Huang, Russel Keathley, Ujin Kim Kim, Horacio Cardenas, Ping Xie, Jianjun Wei, Guangyuan Zhao, Emma L. Barber, Ernst Lengyel, Kenneth P. Nephew, Victoria Bae-Jump, Bin Zhang, Daniela Matei. Comparative analysis of transcriptomic and immunological profiles in treatment-naïve black and non-Hispanic White women with high-grade serous ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7047.

Black women face the highest mortality-to-incidence ratio from high grade serous ovarian cancer (HGSOC). This study investigated biological differences in HGSOC tumors from Black vs. White women. HGSOC from 35 Black and 31 White patients were analyzed by Infinium Methyation-EPIC array and RNA sequencing. 191 CpG sites were differentially methylated (FDR < 0.05, β value change> 10%) and 277 genes were differentially expressed (FDR < 0.05). Gene Ontology identified enriched pathways related to DNA damage response, p53/apoptosis signaling, and cholesterol/lipid metabolism directly connected with genes like INSR, FOXA1 and FOXB1. INSR and FOXA1 knockdown enhanced cisplatin sensitivity and inhibited cell proliferation and colony formation. Tumors from Black patients were infiltrated by fewer CD4+ naïve and regulatory T-cells. Overall, differences in DNA methylation, transcriptomic profiles and immune cell infiltration were detected in tumors from Black vs. White patients. Further investigation is warranted into how these differences may affect treatment response and outcomes in Black women.

Reprogramming of cellular metabolism is a hallmark of cancer. Cancer cells undergo metabolic adaptations to maintain tumorigenicity and survive under the attack of immune cells and chemotherapy in the tumor microenvironment. Metabolic alterations in ovarian cancer in part overlap with findings from other solid tumors and in part reflect unique traits. Altered metabolic pathways not only facilitate ovarian cancer cells' survival and proliferation but also endow them to metastasize, acquire resistance to chemotherapy, maintain cancer stem cell phenotype and escape the effects of anti-tumor immune defense. In this review, we comprehensively review the metabolic signatures of ovarian cancer and their impact on cancer initiation, progression, and resistance to treatment. We highlight novel therapeutic strategies targeting metabolic pathways under development.

Supplemental tables include gene lists related to Figure 5, additional experimental data related to Figure S3I, and primers sequence. Table S1. Numbers of spheroids were counted for each cell dilution; Table S2. List of 20 genes that exhibit a significant change between control and FTO-overexpressing OVCAR5 cells in m6A peak levels, and abundance of the corresponding mRNA transcripta; Table S3. List of primers oligonucleotides; Table S4. Summary of the m6A-seq and RNA-seq; Table S5. List of top 50 hypo-methylation & down-regulated genes; Table S6: List of top 50 hypo-methylation &up-regulated genes.

Additional experimental results supporting the main figures are included. Figure S1. Expression of key RNA methylation regulators in OC; Figure S2. Flow cytometry analysis for ALDH and CD133 in single cell suspensions derived from human ovarian tumors; Figure S3. FTO overexpression in OC cells; Figure S4. FTO overexpression decreases spheroid formation ability of OC cells dissociated from xenografts; Figure S5. m6A activity and tumor initiation capacity; Figure S6. Gene sets enriched in FTO vs. control OC cells; Figure S7. Expression of PDE4B and PDE1C in FTO overexpressed OC cells; Figure S8. The Genome Browser visualizes the predicted motif binding sites for proteins (IGF2BP2 and IGF2BP3) on the input sequence; Figure S9. Effects of phosphodiesterase inhibitors on stemness features are dependent on FTO; Figure S10. Quality control for RNA-seq; Figure S11. Quality control for MeRIP-seq

Supplementary Table S1, S2, S3, and S4

EDITORIAL article Front. Oncol., 01 November 2023Sec. Gynecological Oncology Volume 13 - 2023 | https://doi.org/10.3389/fonc.2023.1325066

Detailed information regarding materials and methods are included in this file.

Abstract Development of resistance to platinum (Pt) in ovarian cancer remains a major clinical challenge. Here we focused on identifying epitranscriptomic modifications linked to Pt resistance. Fat mass and obesity-associated protein (FTO) is a N6-methyladenosine (m6A) RNA demethylase that we recently described as a tumor suppressor in ovarian cancer. We hypothesized that FTO-induced removal of m6A marks regulates the cellular response of ovarian cancer cells to Pt and is linked to the development of resistance. To study the involvement of FTO in the cellular response to Pt, we used ovarian cancer cells in which FTO was knocked down via short hairpin RNA or overexpressed and Pt-resistant (Pt-R) models derived through repeated cycles of exposure to Pt. We found that FTO was significantly downregulated in Pt-R versus sensitive ovarian cancer cells. Forced expression of FTO, but not of mutant FTO, increased sensitivity to Pt in vitro and in vivo (P < 0.05). Increased numbers of γ-H2AX foci, measuring DNA double-strand breaks, and increased apoptosis were observed after exposure to Pt in FTO-overexpressing versus control cells. Through integrated RNA sequencing and MeRIP sequencing, we identified and validated the enzyme nicotinamide N-methyltransferase (NNMT), as a new FTO target linked to Pt response. NNMT was upregulated and demethylated in FTO-overexpressing cells. Treatment with an NNMT inhibitor or NNMT knockdown restored sensitivity to Pt in FTO-overexpressing cells. Our results support a new function for FTO-dependent m6A RNA modifications in regulating the response to Pt through NNMT, a newly identified RNA methylated gene target.

Supplementary Figures S1, S2, S3, S4, S5, S6, S7 and S8

Introduction: Tumor heterogeneity is a key element during the development of chemo-resistance and tumor relapse. It remains unknown how resistant tumor cells influence sensitive cells during conditions of cohabitation. The goal of our study was to determine driving factors mediating interactions between resistant and sensitive cancer cells and the effects of cohabitation on both phenotypes. Materials and methods: We used two isogenic ovarian cancer cell lines pairs, sensitive and resistant to platinum: OVCAR5 (IC50 = 2.05 uM) vs. OVCAR5-CisR (IC50 = 7.26 uM) and PEO1 (IC50 = 5.92 uM) vs. PE04 (IC50 = 14.45 uM) labeled with either GFP or RFP. The cell pairs were seeded together in long direct term culture, separated by fluorescence activated cell sorting (FACS), and used for RNAseq analysis, proliferation assays, Cisplatin IC50 determinations, and cell cycle analysis. Results: Long Term direct co-culture (14 Days) of platinum-sensitive and -resistant cells induced increased proliferation (p < 0.001) and a greater proportions of cells in the S and G2 phases of the cell cycle in sensitive PEO1 and OVCAR5 cells compared with cells cultured by themselves. Resistance to platinum of the sensitive cell lines decreased further by co-culture (IC50 change from 5.92 uM to 2.79 uM in PE01, and from 2.05 uM to 1.51 uM in OVCAR5). No changes in cell proliferation, or resistance to platinum occurred in the platinum resistant cells. Use of conditioned media of resistance cells did not change proliferation, or response to platinum of sensitive cells. RNAseq detected 3840 differentially expressed genes; 1774 upregulated and 2066 downregulated. IPA analysis showed enrichment of the cell cycle pathways (Cell Cycle Control, Cyclins and Cell Cycle Regulation, among others) and upregulation of cell proliferation genes including the E2F1 transcription factor. Western blot and qRT-PCR confirmed upregulation of E2F1 in co-cultured platinum sensitive cells vs cells in monoculture. Furthermore, western blot measurements showed increased levels of other proteins involved in cell cycle regulation such as CDK4 and CDK6. Cell cycle analysis indicated an increase of cells in S and G2 phase in the co-cultured platinum sensitive cells (18.8% up G2 phase; p = 0.031). Treatment with the CDK4/6 inhibitor Palbociclib or with the E2F1 inhibitor HLM006474 reverted the increase in proliferation rate. Conclusion: Long term cohabitation of platinum-sensitive and -resistant cancer cells drives sensitive cells to a higher proliferative state, which is more responsive to platinum. Direct cell-cell contact modulates cell cycle progression in sensitive cells through a mechanism regulated by E2F1. Our data reveal unexpected effects caused by interactions between cancer cells with different proliferative rates and levels of platinum resistance, modelling competition between cells in heterogeneous tumors. Citation Format: Andres Felipe Valdivia, Matthew Cowan, Guangyuan Zhao, Horacio Cardenas, Daniela Matei. E2F1 regulates cell competition, increase proliferation and inhibits chemotherapy resistance in cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2978.

Background: Ovarian cancer (OC) remains one of the deadliest malignancies. Development of resistance to platinum (Pt) is a major clinical problem and understanding its underpinnings will help find new approaches to overcome it. Fat mass and obesity associated protein (FTO) is a N6-methyladenosine (m6A) demethylase and plays an important role regulating how the messenger RNA is processed translating into functional proteins. We recently showed that m6A modifications induced by FTO play a suppressive role in tumorigenicity and survival of OC stem cells. Here we hypothesized that RNA modifications caused by FTO regulate the response of OC cells to Pt. Methods: To study the mechanisms related to FTO implicated in response to Pt; we used OC cells in which FTO was knocked down (KD) via shRNA or overexpressed (OE). Additionally, Pt-resistant (Pt-R) OC cells were obtained through repeated (3-4) exposures to Pt. Cell viability assay determined the IC50 (half maximal inhibitory concentration) to Pt. Pt response in vivo was assessed in FTO expressing vs. FTO KD xenografts. Induction of DNA damage was assessed by immunofluorescence (IF) for γ-H2AX. Apoptosis was evaluated by Annexin V staining in the IncuCyte system. To identify potential targets of FTO-mediated m6A modifications in Pt induced response, RNA-seq and MeRIP-seq were integrated. Results: FTO was significantly downregulated in Pt-R vs. sensitive OC cells. Forced expression of FTO increased sensitivity to Pt in vitro and in vivo, while FTO KD increased Pt resistance (p<0.05). A catalytic mutant FTO did not appreciably alter responsiveness to Pt. Increased γ-H2AX foci and increased apoptosis were observed after exposure to Pt in FTO OE vs. control cells. Through integrated RNA-seq and MeRIP-seq, we identified and validated several potential targets involved in response to Pt including IER5, IER5, ST3Gal, and the enzyme nicotinamide N-methyltransferase (NNMT). NNMT was upregulated and significantly hypomethylated in FTO OE cells and was downregulated in Pt resistant cells. Treatment with an NNMT inhibitor rescued the FTO induced sensitivity to Pt in OC cells demonstrating that its function is necessary in the response to Pt. Conclusions: We identified a new function of FTO-dependent m6A RNA modifications in regulating response to Pt through NNMT, a novel RNA methylated target. Activating FTO could improve response to Pt in OC. Citation Format: Hao Huang, Guangyuan Zhao, Andres Felipe Valdivia, Horacio Cardenas, Yinu Wang, Daniela Daniela Matei. M6A regulated NNMT mediates resistance to platinum in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5359.